Antibiotic 20,798 r.p.

ABSTRACT

The antibiotic 20,798 R.P. of the formula: ##SPC1## 
     Is of special interest because of its anti-tumour activity, being particularly active against acute lymphoblastic and myeloblastic leucaemias. The antibiotic is produced by culture of the microorganism Streptomyces coeruleorubidus (NRRL 3045).

This Application is a continuation-in-part of our application Ser. No.804,088, filed Mar. 4, 1969.

This invention relates to a new antibiotic hereinafter designated 20,798R.P., to a process for its preparation and pharmaceutical compositionscontaining it.

The antibiotic 20,798 R.P. corresponds to the formula: ##SPC2##

This antibiotic is produced alongside antibiotic 9865 R.P., of which thethree principal constituents are designated by the numbers 13,057 R.P.(known by the name daunorubicin), 13,213 R.P. and 13,330 R.P., byculture of the microorganism Streptomyces coeruleorubidus (NRRL 3045)under the conditions described in British Pat. No. 985,598 entitled"Improvements in or relating to Antibiotics and their Preparation"granted to Rhone-Poulenc S.A. on an application filed May 16, 1963,claiming priority from French patent application Ser. No. 898,076applied for on May 18, 1962, or corresponding Belgian Pat. No. 632,391,or in U.S. patent application Ser. No. 280,816 filed on May 16, 1963 byS. Pinnert, L. Ninet and J. Preud'Homme of common assignee. In theaforesaid British patent Streptomyces coeruleorubidus (NRRL 3045) isalso given the synonym "Streptomyces 31723" (NRRL 3045); a specimen ofthe microorganism has been deposited with the United States Departmentof Agriculture, Northern Regional Research Laboratory, at Peoria,Illinois, United States of America, under the number NRRL 3045.Specimens of the microorganism may be obtained freely from the U.S.Department of Agriculture, Agricultural Research Service, FermentationLaboratory, Peoria, Illinois, U.S.A.

The new antibiotic 20,798 R.P. is of very special interest because ofits pronounced anti-tumour activity. It forms acid addition salts. Inthe form of its hydrated hydrochloride, of overall formula C₂₇ H₃₁ O₁₀N,HCl.H₂ O, the antibiotic 20,798 R.P. has the followingphysico-chemical characterisitcs:

Appearance: orange-red needles

Melting point: 225°-230° C.

Solubility: it is soluble in water and alcohols, sparingly soluble inchloroform and practically insoluble in benzene and in diethyl ether.

Ultra-violet spectrum (determined with a solution of 10.09 mg./l. inethanol containing 0.1% N hydrochloric acid): shoulder at about 219 nm(E_(lcm) ^(1%) = 465), shoulder at about 228 nm (E_(lcm) ^(1%) = 635),absorption maximum at 234 nm (E_(lcm) ^(1%) = 782), absorption minimumat 242 nm (E_(lcm) ^(1%) = 483), absorption maximum at 252 nm (E_(1cm)^(1%) = 563), shoulder at about 272 nm (E_(1cm) ^(1%) = 200), absorptionminimum at 280 nm (E_(lcm) ^(1%) = 158), absorption maximum at 290 nm(E_(lcm) ^(1%) = 178), and absorption minimum at about 320 nm(E_(1cm).sup. = 28), where nm is the abbreviation for nanometers.

This spectrum is shown in FIG. 1 of the accompanying drawings in whichthe abscissae give the wave length expressed in nanometers (lower scale)and the wave number in cm.sup.⁻¹ (upper scale), and the ordinate givesthe optical density.

Visible spectrum (determined with a solution of 17.50 mg./l. in ethanolcontaining 0.1% N hydrochloric acid): absorption miximum at 475 nm(E_(lcm) ^(1%) = 143), shoulder at about 533 nm (E_(lcm) ^(1%) = 79) andshoulder at about 553 nm (E_(lcm) ^(1%) = 50).

This spectrum is shown in FIG. 2 in which the abscissae give the wavelength expressed in nanometers (lower scale) and the wave number incm.sup.⁻¹ (upper scale), and the ordinate gives the optical density.

Infra-red spectrum (determined with tablets of a mixture with KBr):

This spectrum is shown in FIG. 3 in which the abscissae give thewavelength expressed in microns (upper scale) and the wave number incm.sup.⁻¹ (lower scale), and the ordinate gives the optical density.

Table 1 below indicates the principal infra-red absorption bands of20,798 R.P. hydrochloride.

                  TABLE I                                                         ______________________________________                                        3410    vs     1540    sh   1195  sh   765   m                                3060    sh     1510    sh   1115  s    750   sh                               2980    s      1500    sh   1080  sh   730   w                                2940    s      1490    sh   1060  s    710   m                                2850    sh     1470    sh   1045  w    690   w                                2580    sh     1460    sh   1030  sh   665   w                                2000    w      1445    s    1005  m    600   m                                1960    w      1430    w    990   vs   570   w                                1720    vw     1410    vs   950   vw   535   w                                1710    w      1380    s    935   w    505   w                                1655    sh     1350    m    915   m    485   m                                1650    sh     1285    vs   905   w    455   m                                1630    sh     1260    vw   870   m    430   m                                1615    s      1255    vw   840   m    390   m                                1580    vs     1235    m    820   m    345   w                                1560    sh     1210    vs   790   m    310   w                                ______________________________________                                         where vs = very strong; s = strong; m = medium; w = weak; vw = very weak      and sh = shoulder.                                                       

Optical rotation: [α]_(D) ²⁰ = + 191 ± 10° (c = 0.2, ethanol containing0.1% N hydrochloric acid).

The antibiotic 20,798 R.P. can be identified by ascending chromatographyon a thin layer of silica gel, using a mixture of benzyl alcohol-ethylformate-formic acid-water (4-4-1-5 by volume) as the solvent. In thissystem 20,798 R.P. has an Rf of 0.57, daunorubicin has an Rf of 0.60 andthe other constituents of antibiotic 9865 R.P. have an Rf above 0.60.

As with daunorubicin, 20,798 R.P. can be hydrolysed in an acid medium toyield an aglycone and an aminated sugar. The aglycone also forms part ofthe invention.

The study of the infra-red spectrum, nuclear magnetic resonance spectrumand mass spectrum enables the following formula II to be attributed tothe aglycone of the antibiotic 20,798 R.P.: ##SPC3##

the formula of the aglycone of daunorubicin being: ##SPC4##

Physically, the aglycone of 20,798 R.P. is distinguished from theaglycone of daunorubicin by the differences in the Rf in chromatographyon thin layers using various solvent systems and in particular:

system 1 : chloroform-dioxan (95-5 by volume)

system 2 : less dense phase of a mixture of ethylacetate-diethylether-M/3 phosphate buffer of pH 4.8 (50-50-25 by volume)

    ______________________________________                                                Aglycone of 20,798 R.P.                                                                         Aglycone of dauno-                                  System  Rf                rubicin Rf                                          ______________________________________                                        1       0.1               0.25                                                2       0.3               0.5                                                 ______________________________________                                    

The aglycone of 20,798 R.P. shows the following physicochemicalcharacteristics:

Appearance: orange-red needles

Melting Point: 285° C.

Solubility: it is sparingly soluble in ethyl acetate, chloroform andmethanol, very sparingly soluble in benzene and practically insoluble inwater, diethyl ether and hexane.

Ultra-violet spectrum (determined with a solution of 10.4 mg/l. inethanol):

absorption maximum at 218 nm (E_(lcm) ^(1%) = 500), shoulder at about228 nm (E_(lcm) ^(1%) = 640), absorption maximum at 233.5 nm (E_(lcm) ¹% = 765), absorption maximum at 251 nm (E_(lcm) ^(1%) = 650) andabsorption maximum at 289 nm (E_(lcm) ^(1%) = 183).

This spectrum is shown in FIG. 4 in which the abscissae give the wavelength expressed in nanometers (lower scale) and the wave number incm.sup.⁻¹ (upper scale), and the ordinate gives the optical density.

Visible spectrum (determined using a solution of 10.4 mg./l. inethanol):

shoulder at about 470 nm (E_(lcm) ^(1%) = 268), absorption maximum at495 nm (E_(lcm) ^(1%) = 284), absorption maximum at 528 nm (E_(lcm)^(1%) = 175) and shoulder at about 570 nm (E_(lcm) ¹ % = 24).

This spectrum is shown in FIG. 5 in which the abscissae give the wavelength expressed in nanometers (lower scale) and the wave number incm.sup.⁻¹ (upper scale), and the ordinate gives the optical density.

Infra-red spectrum (determined with tablets of a mixture with KBr):

This spectrum is shown in FIG. 6 in which the absissae give thewavelength expressed in microns (upper scale) and the wave number incm.sup.⁻¹ (lower scale), and the ordinate gives the optical density.

Table II below indicates the principal infra-red absorption bands ofthis product:

                  TABLE II                                                        ______________________________________                                        3540    sh     1540    w    1065  s    745   w                                3500    sh     1505    w    1060  sh   710   m                                3450    s      1470    sh   1030  m    695   w                                3100    vw     1445    s    990   s    680   vw                               2970    m      1435    m    940   s    660   w                                2935    m      1420    m    915   m    625   sh                               2890    w      1380    s    905   w    615   sh                               2840    w      1350    sh   880   w    590   w                                2650    sh     1285    vs   860   w    545   w                                1655    sh     1250    s    840   vw   500   vw                               1635    sh     1210    s    810   s    485   w                                1615    vs     1190    sh   790   m    450   m                                1585    vs     1110    m    765   m    430   w                                ______________________________________                                         where vs = very strong; s = strong; m = medium, w = weak; vw = very weak      and sh = shoulder.                                                       

The bacteriostatic activity of 20,798 R.P. was determined by one of thedilution methods usually employed for this purpose. For eachmicroorganism the minimum concentration of antibiotic, which underspecified conditions inhibits all visible development of themicroorganism in an appropriate nutrient medium, was determined. Theresults of the various determinations are collected in Table III below,in which the minimum bacteriostatic concentrations are expressed inmicrograms of substance per cc. of test medium.

                                      TABLE III                                   __________________________________________________________________________                                  Minimal in-                                                                   hibitory con-                                   Bacterial strains tested      centration μg/cc.                            __________________________________________________________________________    Staphylococcus aureus, strain 209 P ATCC 6538 P                                                             100                                             Streptococcus faecalis (Faculty of Pharmacy, Paris)                                                         greater than 33                                 Mycobacterium smegmatis ATCC 607                                                                            5.8                                             Klebsiella pneumoniae ATCC 10,031                                                                           greater than 330                                Klebsiella pneumoniae resistant to antibiotic 9865 R.P.                                                     greater than 200                                Streptococcus pyogenes hemolyticus (strain Dig. 7,                            Institut Pasteur)             11                                              Diplococcus pneumoniae (strain Til, Institut Pasteur)                                                       6.1                                             Sarcina lutea ATCC 9341       12                                              Bacillus subtilis (ATCC 6633) 21                                              Mycobacterium phlei (Bacteriological Institute of Lyon)                                                     3.1                                             Mycobacterium para-smegmatis (A 75, Lausanne)                                                               3.1                                             Proteus X 19                  greater than 83                                 Brucella bronchiseptica (CN 387 - Wellcome Institute)                                                       170                                             Pasteurella multocida (A 125, Institut Pasteur)                                                             14                                              Lactobacillus casei ATCC 7469 24                                              Neisseria catarrhalis A 152   greater than 28                                 Salmonella paratyphi A (Lacasse, Institut Pasteur)                                                          greater than 83                                 __________________________________________________________________________

The anti-tumour activities of 20,798 R.P. have been demonstrated in thelaboratory, where the antibiotic has shown itself to be particularlyactive against graftable tumours of mice such as the solid form ofsarcoma 180 and of leucaemia L 1210.

The toxicity of 20,798 R.P. has been studied principally on mice. The50% lethal dose or LD₅₀ was determined intra-peritoneally:

LD₅₀ = 6.5 mg./kg. i.p. The 50% active dose or AD₅₀ was determined onmice by subcutaneous administration in the case of sarcoma 180: AD₅₀ =0.75 mg/kg. s.c., and by intraperitoneal administration in the case ofleucaemia L1210:

    ad.sub.50 = 0.8 mg./kg. i.p.

According to a feature of the invention, a process for the production ofantibiotic 20,798 R.P. comprises cultivating Streptomycescoeruleorubidus (NRRL 3045), or a 20,798 R.P.-producing mutant thereof,in a nutrient medium containing assimilable sources of carbon, nitrogen,and inorganic substances until substantial antibiotic activity isproduced by the said microorganism in the said medium, and separating20,798 R.P. formed during the culture from the medium. The culture ofStreptomyces coeruleorubidus (NRRL 3045) may be carried out, asdescribed in British Pat. No. 985,598, by any method of aerobic surfaceor submerged culture, but the latter is preferred for reasons ofconvenience. For this purpose the various types of apparatus currentlyused in the fermentation industry are employed.

The following scheme for making a production culture is preferably used:##STR1##

The fermentation medium must contain assimilable sources of carbon,nitrogen, and inorganic substances and, optionally, growth factors, allthese ingredients being introduced in the form of well-definedsubstances or as complex mixtures, such as those encountered inbiological products of various origins.

Carbohydrates, such as glucose, lactose, sucrose, molasses, dextrins,starch, and other carbohydrates such as the sugar alcohols, e.g.mannitol, may be used as carbon sources, and also certain organic acids,e.g. lactic, citric, and tartaric acids. Certain animal or vegetableoils such as lard or soya oil may advantageously replace these variouscarbon sources, or be combined with them. A wide range of sources ofassimilable nitrogen are suitable. They may be simple chemicalsubstances such as nitrates, inorganic and organic ammonium salts, ureaand amino acids. They may also be complex substances containing nitrogenprincipally in the form of protein, such as casein, lactalbumin, glutenand hydrolysates thereof, soya-, groundnut-, and fish-meals, meatextracts, yeast, distillers' solubles and corn-steep. Amongst theinorganic substances added, certain may exert a buffering orneutralising effect, such as alkali metal or alkaline earth metalphosphates and calcium and magnesium carbonates. Other inorganicsubstances contribute to the ionic equilibrium necessary for thedevelopment of Streptomyces coeruleorubidus (NRRL 3045) and theformation of the antibiotic, for exmple alkali metal and alkaline earthmetal chlorides and sulphates. In addition, certain substances act asactivators of the metabolic reactions of Streptomyces coeruleorubidus(NRRL 3045); these are the salts of zinc, cobalt, iron, copper andmanganese.

The pH of the nutrient medium at the beginning of culture should bebetween 6.0 and 7.8, and preferably 6.5 to 7.5. The optimal temperaturefor the culture is 25°-28° C., but satisfactory production is obtainedat temperatures between 23° and 35° C. The aeration of the fermentationmedium may be varied over a fairly wide range of values but it has beenfound that aeration rates of 0.3 to 2 liters of air per minute per literof medium are particularly suitable. The maximal yield of antibiotic isobtained after 2 to 5 days of culture, this period depending essentiallyon the medium used. From the foregoing it will be appreciatd that thegeneral conditions for the culture of Streptomyces coeruleorubidus (NRRL3045) for the production of antibiotic 20,798 R.P. may be varied to afairly wide degree.

20,798 R.P. can be isolated from the fermentation broths of Streptomycescoeruleorubidus (NRRL 3045) by various methods. The fermentation brothcan be filtered at a pH of between 1.5 and 9. It is advantageous tocarry out this operation using an acid medium, and particularly oneacidified to a pH between 1.5 and 2 by means of oxalic acid. It is alsopossible to carry out the filtration at a pH between 2 and 7, preferablyabout 2, in the presence of an aliphatic alcohol containing 1 to 3carbon atoms.

After the filtration procedures mentioned above, the mixture ofantibiotics (constituents of 9865 R.P. and 20,798 R.P.) is obtained inaqueous or aqueous-alcoholic solution and it is then extracted with awater-immiscible organic solvent, such as butanol, methyl isobutylketone, ethyl acetate or chloroform, at a pH between 5.5 and 9,preferably about 8.5. This extraction may optionally be preceded by atreatment on an ion exchange resin, in which case the aqueous solutionis adjusted to a pH of about 4 and then fixed onto a cationic carboxylicexchange resin, preferably Amberlite IRC 50, in the acid form. Elutionis then effected with an acid or saline aqueous-alcoholic solution,preferably with methanol containing 10% of water and 1% of sodiumchloride. The eluate is concentrated to remove the alcohol andthereafter extracted as indicated above.

The fermentation broth may also be directly extracted with awater-immiscible organic solvent, such as butanol, ethyl acetate orchloroform, at a pH between 5.5 and 9, preferably about 8.5. In thiscase, all the antibiotic material passes into the organic phase which isseparated from the aqueous phase by the usual processes.

When desired, the fermentation broth can, before the extractionprocedures, be treated with an acid at a temperature between 10° and 75°C. for a period of from 48 hours to 30 minutes, the length of timevarying inversely with the temperature, so as to enrich the antibiotic9865 R.P. with daunorubicin. Under these conditions the antibiotic20,798 R.P. is not hydrolysed. For acidification of the fermentationbroth inorganic acids (hydrochloric, phosphoric or sulphuric acid) ororganic acids (acetic or oxalic acid etc.) are suitable at variousconcentrations, but preferably hydrochloric acid or oxalic acid is usedat concentrations of between 0.01N and 1N. The hydrolysis is preferablycarried out for a period of from 15 hours to 1 hour at a temperature offrom 20 C. to 50° C., the length of time varying inversely with thetemperature.

Whatever the method of extraction chosen, the mixture of antibiotics(constituents of 9865 R.P. and 20,798 R.P.) is finally obtained inorganic solution. It can be advantageous at this stage to carry out apurification by successively transferring the mixture of antibioticsinto aqueous solution and then into organic solution by changing the pH.The mixture of crude antibiotics can then be isolated from the organicsolution obtained in the last-mentioned operation by concentration or byprecipitation with a poor solvent for the antibiotics such as hexane.

A paticularly advantageous method of isolation consists in acidifyingthe organic solution to a pH of about 4, preferably by means of aceticacid, and then concentrating it to a small volume under reducedpressure. The addition of a poor solvent, such as hexane, to theresulting concentrate then causes the precipitation of the mixture ofantibiotics.

The mixture of crude antibiotics can also be isolated by the formationof a salt, such as the hydrochloride, prepared by adding an acid, suchas hydrochloric acid, to the organic solution of the antibiotics andprecipitating the salt of the antibiotics by addition of a poor solventfor them such as acetone.

20,798 R.P. can be separated from the constituents of the antibiotic9,865 R.P. by applying conventional methods such as chromatography onvarious adsorbents, counter-current distribution or partition betweendifferent solvents. The various counter-current distribution procedurescan, of course, be preceded or followed by conventional purificationprocedures, notably by extraction or recrystallisation so as to yield20,798 R.P. in a form which is suitable for the therapeutic applicationenvisaged.

The following Examples illustrate the invention. In the following, thepercentage of 20,798 R.P. is always determined by chromatography on athin layer of silica gel using the solvent system benzyl alcohol-ethylformate-formic acid-water (4-4-1-5 by volume) and direct fluorescimetricmeasurement of the chromatogram relative to a sample of the pureantibiotic.

EXAMPLE 1

A fermentation broth containing 20,798 R.P. is produced by the proceduredescribed in Example 1 of British Pat. No. 985,598 as follows:

A 170 liter fermentation vessel is charged with:

    ______________________________________                                               corn steep  2.400 kg.                                                         sucrose     3.600 kg.                                                         calcium carbonate                                                                         0.900 kg.                                                         ammonium sulphate                                                                         0.240 kg.                                                         water to    100 liters.                                                ______________________________________                                    

This culture medium has a pH of 6.15. It is sterilised by passage ofsteam at 122° C. for 40 minutes. After cooling, the volume of the brothis 120 liters and the pH is 7.20. The medium is then seeded with 200 cc.of a culture in an agitated Erlenmeyer flask of Streptomycescoeruleorubidus (NRRL 3045). The culture is carried out for 27 hours at26°-27° C. with agitation and aeration with sterile air. It is thensuitable for seeding the production culture.

The production culture is carried out in an 800 liter fermentationvessel charged with the following:

    ______________________________________                                        soya flour           20 kg.                                                   distillers' solubles 2.500 kg.                                                starch               10 kg.                                                   soya oil             2.500 liters                                             sodium chloride      5 kg.                                                    water to             465 liters.                                              ______________________________________                                    

The pH of the medium thus obtained is adjusted to 7.20 with concentratedsodium hydroxide solution (400 cc.). The medium is then sterilised bythe passage of steam at 122° C. for 40 minutes. After cooling, thevolume of the broth is 500 liters and the pH is 6.75. It is then seededwith 50 liters of the culture from the 170 liter fermentation vessel.Culture is carried out at 28° C. for 67 hours with agitation andaeration with sterile air. The pH of the medium is then 7.40 and thevolume of the fermentation broth is 520 liters.

Oxalic acid (30 g. per liter) is added to the fermentation broth (400liters) and it is then heated at 50° C. for 1 hour with stirringthroughout. A filtration aid (12 kg.) is added and the mixture at 50° C.is filtered on a filter press and the filter cake washed with water at50° C. The filtrate, having a volume of 475 liters, is cooled to +5° C.The pH is adjusted to 4.5 by addition of concentrated sodium hydroxidesolution. The filtrate is thereafter passed through a column containingAmberlite I RC 50 (15 liters) in the acid form. The filtrate passesthrough the Amberlite bed in an upward direction at a flow rate of 45liters/hour. The column is thereafter washed with water (150 liters) atthe rate of 45 liters/hour, circulating in an upward direction. Thecolumn is then washed with a solution (225 liters) of methanolcontaining 10% of water, circulating in a downward direction, at a flowrate of 45 liters/hour. The washings are discarded and the column iseluted with a solution of the following composition:

    ______________________________________                                        sodium chloride     10      g.,                                               water               100     cc.,                                              methanol to make up to                                                                            1000    cc.                                               ______________________________________                                    

The eluate, which contains the greater part of the antibiotics, has avolume of 120 liters. It is concentrated under reduced pressure at 35°C. to a volume of 10 liters. The concentrate is successively extractedat pH 8.5 with chloroform (2 × 12 liters). The chloroform solution isconcentrated under reduced pressure to a volume of 200 cc.; n-butanol(0.8 liters) is added and the resulting solution again concentratedunder reduced pressure to a volume of 0.4 liters. 2N Hydrochloric acid(20 cc.) is added to this solution with stirring throughout. Theacidified solution is heated to 65° C. and acetone (600 cc.) at 50° C.is added slowly. A mixture of the crystalline hydrochlorides of theantibiotics (15 g.) is obtained.

This mixture contains 20% of 20,798 R.P. and 70% of daunorubicin.

EXAMPLE 2

The mixture of the hydrochlorides of the antibiotics obtained in Example1(15 g.) is dissolved, with stirring, in a mixture of chloroform-water(1--1 by volume; 600 cc.). The pH is adjusted to 8.5 by addition of Nsodium hydroxide solution (18 cc.). This system is subjected to acounter-current distribution of 4 transfers in 1 liter ampoules. Theheavy phase (solvent) is used as the mobile phase and the light phase(aqueous phase) as a stationary phase, and in each case 300 cc. of eachphase are employed. The aqueous phase from ampoule 1 (solution A), thesolvent phase from ampoule 5 (solution B) and the mixture of aqueousphase -- solvent phase from ampoules 2, 3 and 4 are treated separately.

Solution A is successively extracted with a mixture ofchloroform-n-butanol (1--1 by volume; 2 × 150 cc.) at pH 8.5. Theextracts are concentrated under reduced pressure to a volume of 30 cc.The butanol solution thus obtained is adjusted to pH 3 by slowly addingn-butanol saturated with N hydrochloric acid (15 cc.). The hydrochlorideof 20,798 R.P. crystallises. The crystals are filtered off, washed anddried to yield the hydrochloride of 20,798 R.P. (1 g.).

Solution B is concentrated to a volume of 30 cc. and thereafter treatedlike solution A. The hydrochloride of daunorubicin (3.9 g.) is obtained.

The mixture of ampoules 2, 3 and 4 is concentrated to a volume of 200cc. n-Butanol (600 cc.) is added and the solution concentrated to avolume of 100 cc. On addition of hexane (1000 cc.), a mixture (8 g.)containing 20% of 20,798 R.P. and 70% of daunorubicin is obtained.

EXAMPLE 3

The hydrochloride of 20,798 R.P. (2.6 g.) obtained as described inExaample 2 is dissolved in a mixture of dioxan-water (4-1 by volume;31.6 cc.). Anhydrous dioxan (200 cc.) is added slowly, whilst stirringslowly, in order to cause the hydrochloride of 20,798 R.P. tocrystallise. The crystals are filtered off and washed with anhydrousdioxan (10 cc.). The crystals are dried for 15 hours at 50° C. underreduced pressure. The pure hydrochloride of 20,798 R.P. (2.06 g.) isobtained in a yield of 79%. This product has the following elementarycomposition:

    C = 54.8%, H = 6.0%, 0 = 31.9%, N = 2.5%, Cl = 5.8%, H.sub.2 O = 3.0%.

EXAMPLE 4

The product obtained in Example 3 (100 mg.) is dissolved in a mixture ofmethanol-N sulphuric acid (1--1 by volume). The solution is heated for 1hour on a boiling water-bath. On cooling, crystals are obtained whichare filtered off, washed and dried. The aglycone of 20,798 R.P. (36mg.), melting at 285° C., is obtained.

The present invention includes within its scope pharmaceuticalcompositions comprising 20,798 R.P. in association with a compatiblepharmacologically acceptable carrier and/or a compound which may itselfby physiologically active, for example an ntibiotic. These compositionsmay be made up in any phamaceutical form appropriate for parenteraladministration.

Preparations according to the invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or suspending media arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. These compositionsmay also contain adjuvants such as preserving, wetting, emulsifying anddispersing agents. They may be sterilised by, for example, filtrationthrough a bacteria-retaining filter, by incorporation in thecompositions of sterilising agents, by irradiation, or by heating. Theymay also be manufactured in the form of sterile solid compositions,which can be dissolved in sterile water or some other sterile injectablemedium immediately before use.

The percentage of active ingredient in the compositions of the inventionmay be varied, it being necessary that it should constitute a proportionsuch that a suitable dosage for the therapeutic effect desired shall beobtained. Obviously several unit dosage forms may be administered atabout the same time.

In human therapy 20,798 R.P. is active, particularly against acutelymphoblastic and myeloblastic leucaemias, when administeredintravenously at daily doses of between 0.1 and 2 mg./kg. for an adult;a composition for injection for example, consists of a solution of 0.1mg./cc. of active substance in physiological serum.

We claim:
 1. A pharmaceutical composition for parenteral administrationwhich comprises, as active ingredient, an effective proportion of 20,798R.P., of formula; ##SPC5##or a non-toxic acid addition salt thereof, inassociation with enough of an injectable pharmaceutically acceptablecarrier to render said composition suitable for parenteraladministration.
 2. Method of treating acute lymphoblastic ormyeloblastic leucemia which comprises administering intravenously to apatient suffering therefrom between 0.1 and 2 mg./kg. of 20,798 R.P. offormula: ##SPC6##or a non-toxic acid addition salt thereof.